Our long-term goal is to create a suite of robust and quantitative tools and technologies for dissecting proteolytic signaling pathways, and to apply them near-term to prioritize and test the importance of caspase cleavages in apoptosis. There are >500 proteases in humans. However, until recently there were no unbiased proteomic approaches for determining which intact proteins are cleaved in cells or extracts, or the exact site of the cleavage. The current R01 grant (R01 GM081051) allowed us to develop a first generation positive enrichment N-terminomics technology for direct site-specific labeling of free N-termini produced by proteases in cells, extracts, and serum. Key to this technology was the use of an engineered peptide ligase, called subtiligase which has now been distributed to >30 laboratories. Currently we have identified >1400 proteins cleaved by caspases, cysteine-class proteases, that are activated in the apoptotic process and specifically cleave after aspartic acid. This vast data set more than doubles the number of known caspase targets and tells us what can be cut, but not how fast or how conserved in phylogeny. In Specific Aim 1, we will use quantitative selected reaction monitoring (SRM) approaches to determine for the first time for intact proteins in a cell the fundamental rate constants (kcat/KM) for individual caspase substrates. This will allow us to rank them by the catalytic rates at which they are cleaved by specific caspases in human cells and extracts. In Specific Aim 2, we plan to determine how evolutionarily conserved these substrates are through the characterization of caspase cleaved products from primitive to advanced metazoans: worm, fly, mouse and human. We hypothesize that critical nodes are likely those cleaved most rapidly and most highly conserved. In Specific Aim 3, we will test this hypothesis by site-specific proteolysis of selected individual targets. We will use a new genetically encoded highly specific split-TEV protease (the SNIPer) developed in our laboratory, which can be activated by a small molecule and directed to cleave a single target. This will allow us to test if proteolysis of selected nodes is indeed capable of initiating apoptosis similar to how many cancer drugs target an individual caspase substrate. The identification and confirmation of critical nodes in apoptosis will provide insight into the fundamental process of cellular deconstruction and could serve to identify targets for new cancer drugs. Specific Aim 1: Development of the global quantitative methods for kinetic analysis of kcat/KM for intact caspase substrates in extracts and cells Specific Aim 2: Examination of the conservation of caspase cleavage sites and substrates in model metazoans. Specific Aim 3: Site-specific proteolysis of a selected set of conserved and rapidly cleaved substrates using the SNIPer technology.